Compound sinusoidal grid surface with nanometric finish plays a significant role in modern systems and precision calibrator, which can make the systems smaller, the system structure more simple, reduce the cost, and promote the performance of the systems, but it is difficult to design and fabricate by traditional methods. In this paper, a compound freeform surface constructed by a paraboloidal base surface and sinusoidal grid feature surface is designed and machined by slow tool servo (STS) assisted with single point diamond turning (SPDT). A novel combination of the constant angle and constant arc-length method is presented to optimize the cutting tool path. The machining error prediction model is analyzed for fabricating the compound sinusoidal grid surface. A compound sinusoidal grid surface with 0.03 mm amplitude and period of 4 is designed and cutting process is simulated by use of MATLAB software, machining experiment is done on ultra-precision machine tool, the surface profile and topography are measured by Taylor Hobson and Keyence VR-3200, respectively. After dealing with the measurement data of compound freeform surface, form accuracy 4.25 μm in Peak Village value (PV), and surface roughness 89 nm in Ra are obtained for the machined surface. From the theoretical analysis and experimental results, it can be seen that the proposed method is a reasonable choice for fabricating the compound sinusoidal grid surface. When compared with traditional elements, compound freeform surface elements can obviously make the systems smaller, make the system structure much simpler, reduce the cost, and promote the performance of the systems, and it has drawn more attention in recent years ,2. The ca) on binderless tungsten carbide [Nowadays, many researches have concentrated on how to design and manufacture a single simple piece of optical surface, such as spherical or aspherical surface, off-axis surface, and simple micro-structure surface. Ying Cheng et al. manufactured an off-axis optical reflective integrator by ultra-precision machining, the advancement in this study is to reduce the stepwise discontinuity among the facets to make it applicable for turning, and the off-axis optical reflective integrator is more simple in structure and smaller in size when compared with existing refractive integrators, and simulations and experiments show that above 90% of illumination uniformity can be achieved using this new design . Xiaodon carbide . Shijun  carbide . Szymon  carbide . Jayant  carbide . S. Wojc carbide . From th carbide .By analyzing the previous research work, it can been seen that most of research work concentrated on manufacturing single freeform surfaces, few researches investigated on machining compound freeform surfaces. In this paper, a compound freeform surface is designed by combining a paraboloidal base surface and a sinusoidal grid feature surface, optimization of cutting tool path, tool nose radius compensation, and error analysis are done in Compound freeform surface is different from traditional freeform surface in generating, traditional surface is confirmed by an equation or a series of equations, compound freeform surface is obtained by combining double or more freeform surfaces, and From the above illustration of freeform surface, we can reach a conclusion that compound freeform surfaces compose of a base surface and other one feature surface or more feature surfaces, which compares with the traditional single freeform surface. In view of the complex composition of compound freeform surface, the tool path will be more complicated and more challenges will be faced. In this paper, authors select the compound sinusoidal grid surface to be fabricated, which consists of a paraboloidal base surface and a sinusoidal grid feature surface.Compound sinusoidal grid surface include two features, paraboloidal surface and sinusoidal grid surface. In this section, a base surface paraboloidal surface model will be established, the equation of which is described, as follows:This section mainly describes the process of paraboloidal surface establishing, which is the first step to build compound sinusoidal grid surface.From the previous sections, we can know that a compound freeform surface is composed of two or more features; this section will present the second feature surface that is named sinusoidal grid surface, which can be expressed as Equation (2).Base surface and feature surface have been established up to now, and the following research will concentrate on integral compound freeform surface generation. According to According to Equation (3), the calculated compound sinusoidal grid surface consequence is shown in As displayed in In this paper, a compound freeform surface on a cylindrical end face was manufactured, there are two cutting path generation methods including constant angle and constant arc-length methods. As is depicted in For the sake of achieving high accuracy of the compound freeform surface, it is critical to choose an appropriate cutting path. As for the constant angle tool path that is displayed in S cutting path is that each arc between double adjacent cutting points is a constant value, as shown in When compared with constant angle tool path, the main characteristic of constant arc-length ΔDepending on the above analysis, there is a high accuracy at the central region with constant angle cutting path, and the surface accuracy will be superior in outer area with constant arc-length cutting path, so this paper proposed a method that combines the constant angle and the constant arc-length means that the central region of compound sinusoidal grid surface adopts constant angle cutting path and the outer area employs a constant arc-length cutting path. The recombination result of constant angle and constant arc-length method is displayed as As is shown in r, clearance angle α, which is shown as The previous works have determined the compound sinusoidal grid surface, cutting path, and tool contact points. Ultra-precision machining also requires a suitable diamond tool with optimal parameters including tool nose radius Tool geometry parameters have a great influence on the machined surface feature, thus a series of fit tool geometry parameters is essential to guarantee the high accuracy of compound freeform surface finish. The subsequent search is about to concentrate on tool geometry analysis.a shows the way that obtains the surface radial curve for tool nose radius optimum, The first study is about tool nose radius optimum, because the desired compound sinusoidal grid surface is concave-convex severely, and the corresponding cutting path is concave-convex. It will do harm to the compound freeform surface finish accuracy with an unfit tool nose radius. Figure 11Clearance angle is another indispensable factor for optimum of tool geometry, it has a considerable impact on machined surface, as shown in After confirming the advanced double elements, wrap angle is also critical to contribute to surface accuracy, geometry of wrap angle and relation between diamond tool and cutting path are illustrated as Wrap angle The above sections have investigated tool geometry in detail, this part will explain the tool nose radius compensation. Depending on the actually machining processes, diamond tool nose is not a point in fact, but a circular arc, as for that, it is imperative to make tool nose radius compensation to obtain tool location points, the geometry of compensation is presented as The subsequent procedure is to solve the direction of tool nose radius offset, which is expressed as the following equation.The subsequent procedure is to solve the direction of tool nose radius offset, which is expressed as the following equation.r is tool nose radius. All the preparatory works have been accomplished, the last step is tool location points The desired surface compound sinusoidal grid surface can be fabricated on the basis of advanced study, however, there is deviation between the ideal surface and the surface manufactured in reality, including two types of surface errors, named cutting residual error and cutting linearization error. The following section is to analyze them.Cutting residual error is analyzed in the first place, which is caused by the cutting path along feed direction, and it is dominant by tool nose radius and feed rate, schematic diagram of cutting residual error is illustrated in H is cutting residual error, r is tool nose radius and f is feed rate.R is the radius of curvature at the point between adjacent tool trajectory.The cutting residual error including three situations has been explained in above section, and we can restrain the cutting residual error by optimize tool nose radius and feed rate parameters to meet the requirement of accuracy based on Equations (9)–(11), while the tool nose radius is confirmed, feed rate is the critical parameter to ensure the desired surface accuracy. Therefore, in the light of Equations (9)–(11) which can be used to do inverse computation to obtain the optimized feed rate, the corresponding equations of (9)–(11) are as follows.Based on Equation (12), the required feed rate can be expressed as the following:In addition to the cutting residual error, the subsequent section is to analyze cutting linearization error that is the other machining error that is generated in the cutting direction in the course of machining, which is due to the distinction between the ideal surface profile and the linear cutting trajectory, it relies mainly upon the distance between adjacent points along the cutting direction. h is the demand cutting linearization error.From the schematic diagram illustration of cutting linearization error, which uses the approximate concept that an arc between adjacent points is approximate to an ideal arc, therefore, the cutting linearization error is obtained, as follows, based on the approximate concept and the schematic diagram of cutting linearization error.r1 = r2 = 1000, A compound sinusoidal grid surface expressed by Equation (3) with Before manufacturing the workpiece, the critical procedure is to center tool by the UltraSet Optical Tool Setter (USOTS) and to balance the spindle that ensures the machining precision of the desired surface. The applied cutting path shown in In order to verify the effectivity of the proposed method, the compound freeform surface roughness and form accuracy were measurement and evaluated with a precision form measurement system Taylor Hobson  and a high magnification microscope Keyence VR-3200 , respectively.The machining activity was conducted using Nanoform250 ultra-precision machine tool. The actual roughness measurement of the compound sinusoidal grid surface is done in a Taylor Hobson. The surface topography of the compound sinusoidal grid surface measured by Keyence VR-3200 is shown in From the above experiments results, we can know that a compound sinusoidal grid surface is successfully fabricated with well surface finish, which is based on former critical procedures. Above all, the compound freeform surface recombination theory must be acquainted as you expect to machine compound surfaces, because a component with well machined surface needs sufficient theoretical knowledge in order to sustain. Then, an appropriate fabrication method is another vital element, SPDT with STS used in this paper is suitable and highly efficient when compared with the integrated solution (combining fast tool servo with slow tool servo) and laser direct writing method or other solutions, the experiment results have demonstrated that STS is well enough to manufacture compound freeform surfaces. The cutting tool path generation is another indispensable part, most of the research teams acquired failing or defective surfaces on account of inadequate preparation about cutting tool path. A cutting tool path of combining constant angle with constant arc-length path is adopted to process compound sinusoidal grid surface, which contains all the benefits of the two sorts of cutting paths. Finally, a machining error prediction model is necessary to forecast the coming error for designed compound freeform surface, a majority of teams fabricated components by means of trying cutting to guarantee desired surface precision, which is attached with a number of uncertain factors. When compared with the published literature, Xiaodong Zhang et al. studied the mathematic and optical model to propose a grid machining method for the fabrication of compound eye lens. They presented an off-centering machining configuration to avoid the shape distortion that is caused by tool un-alignment. Based on the proposed methods, a compound freeform surface that was constructed by sphere base surface and compound eye lens was successfully machined, but they did not show the surface accuracy and just indicated that the method presented is suitable for the compound eye lens with curve substrate. As far as the author is concerned, there are more and more application fields for compound surfaces with the development of society and technology, and the demand for surface precision will be also greatly improved, so more researches should be done to focus on how to design and machine some compound freeform surfaces.In this paper, the following conclusions can be obtained. Firstly, a new recombination method of compound freeform surfaces is presented that demonstrates the method to achieve a compound freeform surface. Secondly, SPDT assisted with STS is presented in order to successfully manufacture the compound sinusoidal grid surface. Thirdly, a cutting tool path that recombines the constant angle cutting path and the constant arc-length cutting path is proposed to manufacture the compound freeform surface, which can ensure the surface precision. Finally, a fabrication error prediction model is established for forecasting the machining deviation in order to obtain the optimum cutting path parameters, which avoids trying cutting to achieve an ideal surface finish when compared with the other academic research. By cutting an Al6061 workpiece and measuring the machined surface, it is found that the roughness value and PV value of the machined compound freeform surface can be 89 nm and 4.25 μm, respectively. Obviously, the theoretical analysis and experimental results demonstrate that the proposed methods are feasible to machine a compound freeform surface and can improve the machining precision.


Combined stereotactic body radiotherapy (SBRT) for lung lesions and conventional radiotherapy (CRT) for nodal areas may be more effective than CRT alone in patients with locally advanced lung cancer.This study included 21 patients with small primary lung tumors distant from the regional nodal areas. The SBRT dose was 40–60 Gy in 4 fractions. CRT doses were 66 Gy in 30 fractions for non-small cell lung cancer and 52.5 Gy in 25 fractions for small cell lung cancer.The median follow-up duration was 12 months, and the median survival was 13 months. The 1 year overall survival, local recurrence-free survival, and distant metastasis-free survival rates were 60.5, 84.8, and 62.1%, respectively. Two patients experienced in-field local recurrence combined with out-field regional recurrence and/or distant failure. The major recurrence pattern was distant failure . Three patients aged ≥79 years experienced grade ≥ 3 acute radiation pneumonitis, and one also had idiopathic interstitial pneumonia.The combination of SBRT for the lung lesion and CRT for the nodal region seems to be effective and safe for lung malignancies. However, patients older in age and/or with underlying pulmonary disease require stricter lung dose constraints. Definitive concurrent chemoradiotherapy (CCRT) has been recommended as the standard treatment for unresectable or medically inoperable stage II–III non-small cell lung cancer (NSCLC), based on the findings of randomized prospective trials –3. DefinAs the primary lung lesion is located adjacent to the mediastinum and hilar lymph node (LN) region in most lung cancer patients, similar fractionation schemes are generally used for primary lung tumors and metastatic LNs. Some patients, however, are diagnosed with an isolated primary lesion, relatively small in size and distant from the nodal area Fig. . AlthougStereotactic body radiotherapy (SBRT) consists of the administration of one or a few fractions of radiation (with high doses in each fraction), and is expected to achieve improved local control. Several reports indicate the effectiveness of SBRT in the management of patients with early-stage NSCLC, with 2–3 year local control rates of approximately 90% in patients with T1–2 disease –8. In adThis strategy might also reduce toxicities, owing to the reduction in PTV margins and the characteristics of SBRT including high targeting accuracy and rapid dose falloff. Because the SPACE trial found that the rates of pulmonary and esophageal toxicities were lower with SBRT than with CRT , our cenThe records of patients diagnosed with lung malignancy and treated with S + CRT at our hospital between March 2009 and July 2017 were retrospectively reviewed. All patients who received S + CRT had a small isolated primary lesion located distant from the nodal area  reflecting respiratory motion were acquired. The gross tumor volume (GTV) of the lung was delineated at the end-exhale phases using the lung setting . The internal target volume (ITV) was contoured using maximum intensity projection images, whereas the PTV was a 5 mm expansion of the ITV. The SBRT dose  was administered in four fractions, as determined by the radiation oncologist. After SBRT, all patients underwent CT  for three-dimensional CRT planning. The GTV at the LN was contoured using the mediastinal setting , and the PTV margins were maintained as 7 mm radially and 10 mm longitudinally. The clinical target volumes were also delineated for some patients to cover the regional nodal areas in the same axial sections; however, smaller PTV margins  were considered in these patients. In patients treated with induction chemotherapy, the post-chemotherapy volume was defined as the GTV. Standard radiotherapy consisted of 66 Gy (2.2 Gy/fraction) for NSCLC and 52.5 Gy (2.1 Gy/fraction) for SCLC.The organs at risk (OAR) included healthy lung tissues, the esophagus, the spinal cord, and the heart. The normal organ constraints included a maximal dose for the spinal cord of < 50 Gy, a mean lung dose (MLD) of < 20 Gy, a volume of the lung receiving at least 20 Gy (V20) of < 30%, a mean esophagus dose of < 35 Gy, and heart doses of V60 < 1/3, V45 < 2/3, and V40 < 100%.Chest X-rays (CXR) were obtained weekly to monitor changes in tumor volume and any acute toxicities. Treatment was verified by weekly kV imaging guidance, using set-up correction based on carina and bony anatomy.The main concurrent chemotherapy regimen for NSCLC consisted of weekly doses of paclitaxel combined with cisplatin or carboplatin. Patients with very large tumors and a high risk of radiation-related toxicities were initially treated with induction chemotherapy for volume reduction, with a regimen consisting of two cycles of gemcitabine plus cisplatin in sequential schedules every 3 weeks. Chemotherapy for SCLC consisted of intravenous etoposide and cisplatin administered every 3 weeks for four cycles, with radiotherapy initiated along with the third cycle of chemotherapy. Patients with a poor performance status or poor lung function were recommended to undergo radiotherapy alone.All patients underwent weekly complete blood counts and CXR. Patients were routinely followed up by a radiation oncologist and/or medical oncologist, with chest CT and/or CXR performed 1 month after treatment, every 3 months during the first 2 years, and every 6 months thereafter until 5 years after treatment.The primary outcome was 1 year OS rate. Secondary endpoints included 1 year local recurrence-free survival (LRFS) and distant metastasis-free survival (DMFS) rates and toxicities. Toxicities were evaluated using the Common Terminology Criteria for Adverse Events version 4.03. Adverse events that occurred during treatment and within 3 months after CRT were defined as acute toxicities.Follow-up durations were calculated from the date that treatment was discontinued. OS was calculated using the Kaplan-Meier method from the date of treatment discontinuation until death. Clinical and dosimetric factors of patients with and without grade ≥ 3 acute radiation pneumonitis (RP) were compared using Mann-Whitney U-tests. All statistical analyses were performed using SPSS version 21.0.n = 4), IIIA (n = 8), and IIIB (n = 9). All patients had forced expiratory volume in 1 s (FEV1) ≥40% of normal, making them eligible for the NPC 95–01 study [A total of 21 patients were included; their demographic and clinical characteristics are shown in Table n = 6), including idiopathic interstitial pneumonia (IIP) (n = 1), chronic obstructive pulmonary disease (COPD) (n = 1), and patient refusal (n = 1).The median SBRT dose was 54 Gy , which was administered in 4 fractions . Eight patients who received 60 Gy over 4 fractions underwent treatment twice a week, whereas the others were treated daily. Seven patients were treated with SBRT using three-dimensional conformal RT (3D-CRT), whereas 13 received volumetric arc therapy. Non-isocentric technique using a Cyberknife was applied to one patient. Median time interval between the start of SBRT and the start of CRT was 8 days . The median CRT dose was 60 Gy , and the median dose per fraction was 2 Gy . Only one patient received 6 Gy fractions, which is strictly defined as hypofractionated radiotherapy, rather than CRT. Nevertheless, because this study focused on the difference in fractionation regimens administered to the lung and nodal area, this patient was included. Four patients received intensity modulated RT, and the others received 3D-CRT. Nine patients did not receive any chemotherapy due to old age (≥75 years) and comorbidities . Median survival was 13 months . The 1 year OS rate was 60.5% Fig. , whereasTwo patients experienced in-field local recurrence, combined with out-field regional and/or distant failure. The locations of the in-field recurrences were the mediastinal and low cervical nodal areas. Both patients had NSCLC, with one having recurrence after lobectomy. Neither received any chemotherapy. Five patients exhibited out-field regional failure , with three simultaneously diagnosed with distant recurrence. All out-field regional recurrences occurred in nodal areas, not in lung fields, and all of these patients had NSCLC. Four of these patients received definitive treatment, whereas one received salvage treatment. Three did not receive any chemotherapy. The major recurrence pattern was distant failure, which occurred in nine patients . Of these nine patients, only one had SCLC, two received salvage treatment, and two did not receive chemotherapy.Nine patients experienced acute radiation esophagitis, including eight with grade 2 disease and one with grade 3 disease. Except for one patient who finally progressed to grade 3 late radiation esophagitis, the other patients were successfully managed medically. Eight patients experienced acute RP, including five with grade 2, one with grade 3, and two with grade 4 (life-threatening) disease. The characteristics of the three patients with acute RP of grade ≥ 3 are summarized in Table One patient who initially had IIP showed poor lung function with a DLco of 37%; this patient was treated with hypofractionated radiotherapy to the nodal area (60 Gy over 10 fractions). Nevertheless, as the PTV of the nodal area was very small (27.7 cc), more conservative dose constraints were applied. Dosimetric parameters showed that his MLD was 7.1 Gy and his V20 was 9.1% (Table p = 0.011). Other demographic and clinical factors, including sex, performance status, smoking history, pre-treatment pulmonary function test results, aim of radiotherapy , and combined chemotherapy, were not significantly associated with grade ≥ 3 RP. Due to the small number of patients enrolled, we could not reliably determine the dosimetric parameters, such as MLD, V5, V20, mean ipsilateral lung dose, and PTV size, predictive of grade ≥ 3 RP. Two patients without a history of acute RP experienced late RP of grade 2. None of the patients experienced any grade ≥ 2 hematologic toxicity.The only significant factor associated with grade ≥ 3 RP was older age  and OS  in the SBRT arm [Only two of our patients experienced in-field recurrences at nodal areas alone. A previous study investigated the patterns of loco-regional failure in locally advanced NSCLC patients who received definitive CCRT (66 Gy in 24 fractions) . There wSBRT arm . AlthougSBRT arm . This fiAttempts have been made to administer SBRT to patients with locally advanced cancers. For example, a prospective, single-institution study evaluated the feasibility of CRT (59.4 Gy in 33 fractions) followed by SBRT (20 Gy in 2 fractions or 19.5 Gy in 3 fractions) in patients with stage II–III NSCLC and residual disease on positron emission tomography . Over a Patients administered S + CRT are likely to experience the abscopal effect of SBRT . Becausep = 0.006) and a lower rate of RP  in the SBRT than in the CRT group [Previous studies of patients with NSCLC treated with 63–66 Gy in fractions of 1.8–2 Gy reported grade ≥ 3 pulmonary toxicities presenting as acute complications in 4–5% of patients and late toxicities in 11% . AcceptaRT group , our patOne patient in the present study with grade 4 RP had IIP and a DLco of only 37%. A more pronounced decrease in lung function has been reported in individuals with an initial DLco of ≤50% . Lung coThis study had several limitations, including its retrospective design, the small number of patients, and the heterogeneity of the enrolled patients. Although CCRT was the standard treatment, 43% of these patients did not receive any chemotherapy. Therefore, the results of the present study are not representative of standard populations. The potential numbers of candidates for S + CRT are limited, because relatively few patients present with primary lung lesions distinct from nodal areas.This retrospective analysis of lung cancer patients treated with S + CRT showed promising local control and survival rates, as well as acceptable toxicities. Stricter lung dose constraints are need for older aged patients and those with underlying pulmonary diseases. Additional studies, in larger numbers of patients and with stricter enrollment criteria, are required to confirm these findings.


UV exposure is associated with oxidative stress and is the primary factor in skin photoaging. UV-induced reactive oxygen species (ROS) cause the up-regulation of metalloproteinase (MMPs) and the degradation of dermal collagen and elastic fibers. Garlic and its components have been reported to exert antioxidative effects. The present study investigated the protective effect of garlic on UV-induced photoaging and MMPs regulation in hairless mice. Garlic was supplemented in the diet, and Skh-1 hairless mice were exposed to UV irradiation five days/week for eight weeks. Mice were divided into four groups; Non-UV, UV-irradiated control, UV+1% garlic powder diet group, and UV+2% garlic powder diet group. Chronic UV irradiation induced rough wrinkling of the skin with hyperkeratosis, and administration of garlic diminished the coarse wrinkle formation. UV-induced dorsal skin and epidermal thickness were also ameliorated by garlic supplementation. ROS generation, skin and serum malondialdehyde levels were significantly increased by UV exposure and were ameliorated by garlic administration although the effects were not dose-dependent. Antioxidant enzymes such as superoxide dismutase and catalase activities in skin tissues were markedly reduced by UV irradiation and garlic treatment increased these enzyme activities. UV-induced MMP-1 and MMP-2 protein levels were suppressed by garlic administration. Furthermore, garlic supplementation prevented the UV-induced increase of MMP-1 mRNA expression and the UV-induced decrease of procollagen mRNA expression. These results suggest that garlic may be effective for preventing skin photoaging accelerated by UV irradiation through the antioxidative system and MMP regulation. Wrinkleteration .Excessive exposure to UV irradiation generates reactive oxygen species (ROS) in the skin , and oveROS lead to the activation of transcription factors which causes up-regulation of metalloproteinase (MMPs) and degradation of dermal collagen and elastic fibers . MMP-1 aGarlic has long been used widely not only as a flavoring agent but also as a folk medication and is one of the most well-known herbal medicines worldwide. Numerous therapeutic effects of garlic and its constituents such as antioxidant, anti-microbial, anti-atherosclerotic, anti-diabetic, anti-mutagenic, anti-carcinogenic and immune-modulation activities have been reported ,13,14,15The changes of body weight and serum biochemical indicators in the hairless mice were determined to examine the animal toxicity of garlic powder. Mean body weights of all experimental groups were similarly increased throughout the period of study. There were no significant differences in body weight and food intake between the groups during the eight weeks of the experimental periods (data not shown), suggesting that body weight gain and food consumption were not affected by UV irradiation and garlic supplementation.The hepatotoxicity of garlic was determined by measuring the serum aspartate transaminase (AST) and alanine transaminase(ALT) concentrations . UV irraThe effect of garlic on UV-induced wrinkle formation was observed by visual assessment of close-up photos in the dorsal skins of hairless mice. Chronic UV irradiation induced deep, irregular wrinkling of the skin with hyperkeratosis, and marked reduction in UV-induced wrinkle formation was observed in the UV+G-1% and UV+G-2% groups compared with the UV group A.Chronic UV irradiation induces skin inflammation, which causes excessive epidermal proliferation and skin thickening . The effp < 0.05). The results again showed that 1% garlic supplementation exhibited better protection from UV-induced skin damage.Sections of dorsal skin from hairless mice were stained with Hematoxylin and eosin (H & E) or the Verhoeff van-Gieson method to determine epidermal hyperplasia and elastic fiber, respectively. Epidermal thickness, as a histological feature of photoaged skin, can be used as one of the quantitative parameters to reflect UV-induced skin damage, since thickened epidermis can contribute to skin roughness . Based oElastic fibers are important components of the skin and are responsible for skin elasticity. To visualize the changes in elastic fibers in the dermal areas, histological sections of the skin were subjected to Verhoeff’s staining. As shown in Oxidative stress is a primary factor in the photoaging process. UV increases levels of hydrogen peroxides and other reactive oxygen species in skin and decreases the levels of antioxidant enzymes ,6. IncreMalondialdehyde (MDA) is the aldehydic decomposition product of lipid oxidation, which is the major thiobarbituric acid reactive substance (TBARS). The lipid peroxide levels in skin and serum were determined by measuring the TBARS concentration. As shown in Serum SOD and catalase activities were significantly reduced by UV irradiation, while dietary garlic supplementation significantly increased these enzyme activities compared with the UV-irradiated control group . No signMMPs  have been known to play important roles in the degradation of skin collagen and elastin. To investigate the mechanism underlying the protective effect of garlic on UV-induced photoaging, expressions of MMP-1, MMP-2, and procollagen type 1 were analyzed . The resType 1 collagen is the most abundant collagen in skin connective tissue as well as the human body, and newly synthesized procollagen type 1 is secreted into the dermal extracellular space, and has an anti-wrinkle effect in UV-irradiated skin. Also, a decrease in procollagen expression caused by repeated UV irradiation has been considered to be a cause of photoaging . TherefoS-allylcysteine and allixin, suppress LDL oxidation in vitro. Furthermore, short-term supplementation of garlic in human subjects has demonstrated an increased resistance of LDL to oxidation as well as blood antioxidant parameters [One of the most important biochemical properties of garlic is its antioxidant potential. Garlic or garlic oil supplementation prevented the increase in oxidative stress associated with gentamicin-induced nephrotoxicity or nicotine-induced lipid peroxidation in rats ,20. Oxidrameters ,14.Chronic exposure to UV irradiation results in photoaging of skin including various characteristic changes, such as DNA damage ,21,22, hSkin consists of three layers: the epidermis, the dermis, and the subcutis. The outer layer, the epidermis, is composed of keratinocytes, melanocytes, and langerhans cells. This layer serves as a barrier to protect the body by synthesis of the protein keratin. The dermis is made up of collagen fiber, elastin fiber, and the derma matrix, which are produced by fibroblasts . The epiet al. [Photo-oxidative damage caused by chronic UV exposure is the leading cause of extrinsic aging of the skin. In normal cells, antioxidative defense systems, including SOD, catalase, and glutathione peroxidase, have evolved to quench ROS ,6,24. Hoet al.  demonstret al. . In the in vivo [Photoaging is characterized by the degradation of collagen and the accumulation of abnormal elastin in the dermis, and several MMPs have been implicated in this process ,9,29. Inin vivo . Collagein vivo . SimilarS-allylcysteine (SAC), and vinyl dithiines [Garlic contains unique organosulfur compounds, which provide its characteristic flavor/odor and most of its potent biological activity. The main sulfur compound in both raw garlic and garlic powder is alliin, and allicin was found to be a major constituent of solvent extracted garlic . Allicinithiines .S-alk(en)yl-l-cysteine sulfoxides have been reported [et al. reported that topical treatment of S-allyl cysteine, the most abundant component of aged garlic, exhibits antioxidative, anti-wrinkle activity, and down-regulates MMP-3, -9, and -12 protein expression [et al. [Antioxidative effects of sulfur compounds such as allicin, allyl disulfide, allyl cycteine, alliumthiosulfinates and reported ,33,34. Kpression . Howeverpression . Recentl [et al.  reportedN-acetyl-S-(2-carboxypropyl)-cysteine, N-acetylcysteine and hexahydrohippuric acid, have been detected in human urine after ingestion of garlic [Bioavailability of active ingredients in garlic is likely essential, and it was suggested that the metabolites of allicin, rather than the allicin itself, are responsible for the health effects of garlic. SAC is one of the water-soluble organosulfur compounds in garlic. SAC absorbed rapidly and showed almost 100% bioavailability after oral administration . Also, tf garlic . At presf garlic . Howeverw/w), and 1% garlic supplementation exhibited better or similar effect to 2% garlic supplementation, suggesting that 1% garlic supplementation reached the peak effective dose. The 1% garlic supplementation in the present study was 250 mg/kg/day garlic powder (food intake was about 5 g/day/mice) and was equivalent to approximately 4.5 g~5.7 g garlic powder/day for humans on the bases of energy requirements . Pitana et al. [The doses of garlic supplemented in the present study were 1% and 2% of the diet , purchased from Seosan  in July 2009, were peeled, vacuum dried, and powdered.Fresh garlic (w/w) in commercial mouse diet . Body weight and food intake were regularly measured throughout the study. The animal protocol used in this study was reviewed and approved by the Kyunghee University Institutional Animal Care and Use Committee (KUIACU).Six-week-old female albino hairless mice (Skh:HR-1) were obtained from Central Lab Animals, Inc. . The animals were housed under controlled temperature (23 ± 2 °C), humidity (55% ± 5%) and light (12 h light/dark cycles). All animals had free access to water and commercial pellet food. After an acclimation period (one week), mice were randomly divided into four groups of eight animals: Non-UV, UV-irradiated control, UV+G-1% (UV+1% garlic powder diet group), UV+G-2% (UV+2% garlic powder diet group). Garlic powder was supplemented  at a distance of 20 cm from the UV light source provided by an array of UVA and UVB  lamp with a dose output ratio at 1:4.2 for the first week, with an increase to 55 mJ/cm2 at second week, 63 mJ/cm2 at third week, 68 mJ/cm2 at fourth week, 76 mJ/cm2 at fifth week, 81 mJ/cm2 at sixth week, and 89 mJ/cm2 at seventh to eighth week for total dose of 2850 mJ/cm2. The non-irradiated control group was treated identically with the lamp power off. The integrated UV irradiation was measured with a UV meter . The animals were sacrificed three days after the final irradiation to allow the recovery from the acute UV effects. The blood collected via cardiac puncture was allowed to clot, and the serum was obtained by centrifugation at 3000× g for 30 min. Aliquots of serum samples were stored at −20 °C until analyzed.The mice were exposed to UV irradiation five days per week starting with 50 mJ/cmSkin thickness was assessed by measuring skin-fold thickness. Briefly, midline dorsal skin of mouse was lifted up at the neck and base of the tail by pinching and skin-fold thickness was measured mid-way between the shoulders and hips using a caliper . The graders were blinded to the groups of the mice.The hepatotoxicity of garlic powder was determined by measuring the activities of alanine transaminase (ALT) and aspartate transaminase (AST) using commercial kits .Morphologic changes of mouse skin surface were observed using a digital microscope .For histopathological assessment, the dorsal skin tissue was fixed in 10% neutral buffered formaldehyde solution, embedded in paraffin and sliced into 5 μm sections. H & E staining was performed to assess epidermal hyperplasia which was measured by epidermal thickness. Verhoeff’s staining was performed using a Verhoeff Van Gieson elastin staining kit  to examine elastic fibers.g for 20 min at 4 °C, and total supernatant was used for the analysis.Skin samples were harvested from the sacrificed mice and the skin tissue (0.4 g) was homogenized  with Ultra Turrax  in nine volumes of cold normal saline (4 °C) to obtain the skin tissue homogenate. The un-dissolved pellet was removed by centrifugation at 3000× A fluorometric assay was used to determine ROS level. Non-fluorescent 2’,7’-dichloro fluorescin diacetate (DCFDA) is oxidized to the highly fluorescent 2′,7′-dichlorofluorescin (DCF) in the presence of esterase and ROS, including lipid peroxides . Brieflyw/v TCA and 0.375% w/v TBA in 0.25 N HCl) reagent was added and heated for 15 min, flocculent precipitate was removed by centrifugation at 1000× g for 10 min and absorbance was measured at a 535 nm against a blank containing no tissue homogenate. The MDA concentration was calculated using an extinction coefficient of 1.56 × 105/m·cm.MDA has been identified as the product of lipid peroxidation. MDA levels in the skin and serum were estimated by the thiobarbituric acid assay. To 1 mL of skin tissue homogenate or serum sample, 2 mL TCA-TBA-HCl . Catalase activity was measured according to the method of Aebi . One uniThe production of MMP-1 and MMP-2 were determined in skin homogenate supernatant using commercially available ELISA kits .® Protect Mini kit  to measure the mRNA expression of procollagen I and MMP-1. Complementary DNA was synthesized from mRNA using QuantiTect® Reverse Transcription kit (Qiagen). Real-time PCR was performed using Quanti Tect TM SYBR® Green PCR kit (Qiagen) according to the manufacturer's protocol. The cDNA was amplified for 45 cycles of denaturation (95 °C for 30 s), annealing (58 °C for 30 s), and extension (72 °C for 45 s) using the following primers: GAPDH forward 5′-CAT GGC CTT CCG TGT TCC TA-3′, reverse 5′-GCG GCACGT CAG ATC CA-3′; MMP-1 forward 5′-TTG CCC AGAGAA AAG CTT CAG-3′, reverse 5′-TAG CAG CCC AGA GAAGCA ACA-3′; procollagen type I forward 5′-GGACCT TGG AAG CCT TGG GGA CC-3′, reverse 5′-GGA ATT CGA AACCAC CGG CGT CGA AGG A-3′. Analyses were performed using Rotor-Gene Q® (Qiagen) and gene expression values were calculated based on the comparative ΔΔCT method.Dorsal skin tissues were homogenized with buffer RLT  in ice bath. Total RNA was extracted from dorsal skin tissue lysate using RNeasyp < 0.05.Data were presented as mean ± SD. One-way ANOVA was used to determine treatment effects. Differences among means were inspected using Tukey test and were considered to be significant at All together, the findings of this study indicated that dietary supplementation of garlic (1%) is effective in wrinkle improvement by alleviating oxidative stress and dermal extracellular matrix damage, resulting in the improvement of dermal collagen and elastic fiber status in the hairless mice photoaged model. In conclusion, this study suggests a high possibility for the practical use of garlic as an anti-wrinkle agent.


GATA4) and Friend of GATA 2 protein  form a heterodimer complex that has been shown to influence transcription of genes in a number of developmental systems. Recent evidence has also shown these genes play a role in gonadal sexual differentiation in humans. Previously we identified four variants in GATA4 and an unexpectedly large number of variants in ZFPM2 in a cohort of individuals with 46,XY Differences/Disorders of Sex Development (DSD) .GATA‐binding protein 4  and variant protein–protein interactions.Here, we review variant curation and test the functional activity of GATA4 and ZFPM2 variants we identified are benign in their contribution to 46,XY DSD. Indeed, only one variant, in the conserved N‐terminal zinc finger of GATA4, was considered pathogenic, with functional analysis confirming differences in its ability to regulate Sox9 and AMH and in protein interaction with ZFPM2.Our findings support that the majority of GATA4 and ZFPM2/FOG2 are not causative.Our study helps define the genetic factors contributing to 46,XY DSD and suggests that the majority of variants we identified in  GATA4 and FOG2. Variant curation and functional analysis revealed the majority of these variants are not likely to be causative.We identified a number of 46,XY DSD individuals with variants in  AVSDAtrial ventricular septal defectsCHDcongenital heart diseaseDIH3diaphragmatic hernia 3DSDdifferences/disorders of sex developmentHEK293human embryonic kidney 293OMIMonline mendelian inheritance in manSNVsingle nucleotide variantSRXY946XY sex reversal 9TADtransactivation domainTOFtetralogy of fallot1GATA4 family member has been shown to be involved in development of the heart, pancreas, liver, foregut as well as in the genital ridge of both sexes  were first identified in patients with congenital heart disease (CHD) including Atrial ventricular septal defects (AVSD)  are an evolutionally conserved family that plays various roles in embryonic development. The GATA members have two zinc finger domains that are required for recognition and affinity for DNA and interaction with other transcription factors  directly interacts with GATA4, by forming a heterodimer that represses expression of GATA4 target genes  and a heterozygous change c.779G > A (p.R260Q) . Here we have re‐curated these GATA4 and ZFPM2 variants using updated tools, and have tested their molecular activity in the context of gonadal signaling using several in vitro assays.While screening 279 46,XY DSD individuals for variants in genes known to cause DSD, we previously identified four variants in 22.1GATA4 (MIM# 600576) (four variants in seven patients), detailed in Table GATA4 variants, five presented with hypospadias . Case 5 in addition to hypospadias presented with multiple congenital anomalies including imperforate anus and dysmorphic facial features , c.1180C > A (p.Pro394Thr), and c.1220C > A (p.Pro407Gln), in association with Atrio ventricular septal defect   was located in the N‐terminal Zn‐finger  were located in the C‐terminal TAD  variants in 10 patients occurring in a wide spectrum of 46,XY DSD phenotypes ranging from males with hypospadias , ambiguous genitalia  to 46,XY individuals presenting with female external genitalia  , diaphragmatic hernia , or 46, XY DSD . Interestingly, two of the patients (case 8 and 9) each had a novel variant and an identical second hit in a more commonly observed variant  that has been reported in ClinVar with numerous ZFPM2‐associated conditions. In these cases it is not known whether the variants effect the same or different alleles  of the ZFPM2 amino acid changes occur in annotated functional domains. The ZFPM2 (FOG2) protein has eight Zn‐finger domains, and an N‐terminal region (1–247) that is required for GATA4‐mediated repression of target genes  were reclassified as likely benign or benign. Primarily, the global MAF or Popmax filtering AF (95% CI) in GnomAD was higher than the expected frequency of the condition  was curated as pathogenic. The variant is present in a highly conserved domain without benign variation and absent from population databases. Additional pathogenic supporting evidence was confirmed by familial segregation of the variant in the affected maternal uncle and genotype–phenotype correlation . While three missense  and one in frame deletion (p.L607del) were re‐classified as variants of unknown significance (VUS) due to conflicting or lack of evidence to support classification. Similarly, to individuals with GATA4 variants, limited patient information was available for curation  (benign classification) and case 4 had a well‐described pathogenic variant in the ligand binding domain of the androgen receptor (AR:NM_000044.4:exon7:c.2599G > A (p.V867M))  and case 16: NR5A1:NM_004959.4:c.251G > A (p.R84H)   in NR5A1 activation of mTesco (p < .0001)  using the previously described deleterious variant GATA4 p.G221R, as a positive control  with wild‐type ZFPM2 had a loss of repression of NR5A1 mediated activation of mTesco (comparable to NR5A1/ZFPM2 wild‐type levels without GATA4) (p < .0001)  had a 50% reduction in activity. The other GATA4 variants show activity comparable to wild‐type  of interest was overexpressed in combination with the wild‐type binding partner (ZFPM2) or vice versa in HEK293 cells. The complex was immunoprecipitated using the tag antibody of the wild‐type protein and variant interaction was assessed using the native antibody on Western blot. Wild‐type GATA4 and ZFPM2 protein complex was detected  showed reduced interaction . Zinc finger variants p.G221R and p.W228C variants were almost undetectable  were from distinct ethnic backgrounds such as Pakistan, Cambodia and Indonesia. GnomAD has limited population data for these geographical subsets. Due to the small numbers available from South East Asian countries (like Cambodia and Indonesia), these populations were likely included in the “other” category  which was considered pathogenic. Multiple lines of evidence contributed to pathogenicity including, confirmation of inheritance in an affected family member, genotype–phenotype correlation with a published study  were found in three of cases presented here .These results confirm the outcome of our re‐curation, where the remaining Taken together, our study highlights the importance of periodically reassessing DSD gene variants with up to date curation evidence, and disease/tissue‐specific functional assays, particularly when they have been identified as pathogenic in association with another condition. We believe that an integrative approach between the clinical and research setting is essential to further advance our understanding of the genetic basis of DSD.44.1Collaborating clinicians recruited patients for the study, informed consent was obtained and EDTA bloods were collected. Approval for this study was obtained from the Human Ethics Committee of the Faculty of Medicine at the Royal Children's Hospital (application HREC 22073). DNA was extracted by an independent laboratory such as Victorian Clinical Genetics Service (VCGS) or by other hospital providers.4.2https://www.mutalyzer.nl/). Variants of lower quality were verified by Sanger sequencing (details not shown). The sequencing data for each patient is available from the Sequencing Read Archive (SRA) using reference numbers SRP092281 and project PRJNA350857     and GATA4  were created by site‐directed mutagenesis  according to the manufacturer's instructions  and pCMV6‐Entry‐hZFPM2  both with a C‐terminal Myc‐DDK tag. Prior to introducing mutations to the pCMV6‐Entry‐hGATA4 the vector was modified to remove the Myc‐DDK tag and introduce a C‐terminal HA tag. Sanger sequencing using universal vector primers was used to confirm the introduction of the correct variant.The variant overexpression vectors for 4.5mTesco) and human proximal promoter of AMH  were setup using HEK293‐T human embryonic kidney cells and Lipofectamine‐2000 as the transfection reagent. Cells were setup in 96‐well plates, co‐transfected with promoter vector (75 ng pGL4‐mTesco or 100 ng pGL4‐hAMH) and 5 ng of Renilla (pRL‐TK) as a marker of transfection efficiency. For the mTesco assay: transcription factors were also transfected with or without 40 ng of wild‐type pCMV6‐Entry‐hNR5A1 ; 40 ng of pCMV6‐GATA4 wild‐type  or mutant vectors; and 40 ng pCMV6‐ZFPM2 wild‐type  or mutant vector. Total DNA for each mTesco assay was 200 ng, pCMV‐empty was used to adjust the total DNA for each well. In the case of the hAMH assay: 45 ng wild‐type pcDNA‐WT1 KTS−/−; with or without) 15 ng pCMV‐GATA4 (wild‐type or mutant); and 15 ng pCMV‐ZFPM2 (wild‐type or mutant), were used at a respective ratio of 3:1:1 (WT1:GATA4:ZFPM2). Total DNA for each hAMH assay was 180 ng, pCMV‐empty was used to adjust the total DNA for each well. Assays were lysed 24 hr post‐transfection and luciferase activity was measured using the dual‐luciferase reporter assay  on an Infinite M200 Pro plate reader (Tecan). Each data point represents the average ratio of firefly to renilla luciferase for each condition (performed in triplicate), normalized to the empty vector control (fold change relative to the negative control). The standard error of the mean is shown for three to four independent experiments that were run in technical triplicate.Luciferase transactivation assays for mouse enhancer of Tesco (pGL4‐4.6GATA4 (wild‐type or mutant) over expression, rabbit polyclonal anti‐GATA4 antibody at 1:200 ; and for ZFPM2 (wild‐type or mutant) over expression, mouse monoclonal anti‐FOG2 (H5) at 1:1000 . The next day, cells were washed several times with PBS before incubating with the following secondary antibodies in 1% BSA in PBS: for GATA4 overexpression staining secondary antibodies donkey anti‐rabbit Alexa‐488  and for ZFPM2 overexpression donkey anti‐mouse Alexa‐488 . Nuclear counterstaining with DAPI (blue) was also performed. Images were acquired on a Zeiss AXIO Imager M1 for each overexpressed gene the variants were captured using the same settings as for the wild‐type image.Protein for immunofluorescent imaging was prepared by seeding HEK293‐T cells on 8‐well chamber slides, 200 ng of individual overexpression constructs was transfected with Lipofectamine 2000 (Invitrogen). After 24 hr post‐transfection cells were processed for staining by removing media, briefly washing cells with ice‐cold PBS, fixing cells for 10 min with 4% PFA, 10 min permeabilization with 1% triton‐X‐100 in PBS, and blocking with 2% BSA in PBS. Cells were then incubated overnight with primary antibody in 1% BSA PBS as follows: for 5 HEK293‐T cells in a 24‐well plate and transfecting 800 ng pCMV6‐Entry‐GATA4 (NM_002052.4)  or pCMV6‐Entry‐ZFPM2 (NM_012082.3) overexpression construct  (wild‐type or mutant) with Lipofectamine 2000 (Invitrogen). Protein was harvested 24 hr post‐transfection by removing media and washing the cells with ice‐cold PBS, and lysed using Pierce IP lysis buffer  with protease inhibitors . Protein was quantified using the Pierce BCA protein assay kit , along with BSA protein standards. Five micrograms of total protein was run on a 10% Bis‐Tris gel with MOPS buffer (GATA4), or 4%–12% Bis‐Tris with MOPS buffer (Invitrogen) (ZFPM2), transferred to PVDF membrane, blocked using 5% skim milk powder/TBST and incubated with rabbit polyclonal anti‐GATA4 antibody ; or mouse monoclonal anti‐FOG2 (H5)  overnight at 4°C. After washing with TBST, swine anti‐rabbit HRP (1:10000 DAKO P0399) was incubated at room temperature for 2 hr. After blot washing the Amersham ECL Prime Western blotting detection reagent was used and visualized with the GE Healthcare Life Sciences ImageQuant LAS 4000. Blots were washed and then incubated with loading control—anti‐alpha Tubulin (HRP)  or anti‐GAPDH (HRP) , washed and detected as mentioned above.Protein analysis using Western blot was prepared by seeding 4 × 104.7GATA4 and ZFPM2 proteins was detected as follows: HEK293‐T cells were seeded at 1.6 × 106 in 6‐well plates, transfected with 1.5 ug of pCMV6‐GATA (HA‐tag)  containing the human cDNA ORF of GATA4 (NM_002052.4) (wild‐type or variant); and 1.5 ug pCMV6‐Entry‐hZFPM2 (myc‐DDK tag)  containing the human cDNA ORF of ZFPM2 (NM_012082.3) (wild‐type or variant) with Lipofectamine 2000. After 24 hr, cells were washed and harvested in 500 ul of Pierce IP lysis solution with protease inhibitors , protein lysates were quantitated using the Pierce protein assay kit. For each IP reaction 500 ug of protein lysate  was incubated with 1.5 ul rabbit anti‐HA  (when testing for ZFPM2 protein interaction) or 1.5 ul rabbit anti‐FLAG  (when testing for GATA4 protein interaction) overnight on a suspension mixer with inversion at 4°C degrees. The following day, 20 ul A/G plus agarose beads  was directly added to each IP reaction and incubated for 4 hr on a suspension mixer at 4°C degrees. The IP reaction was then centrifuged at 587g for 5 min at 4°C degrees, supernatant carefully removed and the pellet washed 4–5 times with 500 ul PBS, in the same manner as mentioned above. The IP protein was then resuspended directly in 40 ul 2x SDS protein loading buffer , boiled at 95–100°C for 5 min to denature the protein and release it from the beads. Western blot analysis of the co‐immunoprecipitation was setup as described above: for each IP reaction 15 ul was run per lane; while the 5% of the input sample was run on a separate gel as a control. When testing for GATA4 mutant protein interactions, anti‐GATA4 was used to probe the IP reactions and anti‐ZFPM2 was used to probe the input (as mentioned above). When testing for ZFPM2 mutant protein interactions, anti‐ZFPM2 was used to probe the IP reactions and anti‐GATA4 was used to probe the input (as mentioned above).Interaction of Dr Davis reports having received honoraria from Besins Healthcare and Pfizer Australia and has been a consultant to Mayne Pharmaceuticals, Lawley Pharmaceuticals and Que Oncology. All other authors declare no conflict of interest.The manuscript was compiled and written by J.vdB, K.A and A.S. Patient database, sample preparation, sequencing and analysis was performed by J.vdB, G.R, S.E and K.A. In vitro and functional studies were performed by J.vdB, while supervision and project guidance were provided by K.A and A.S. Management and recruitment of patients involved in the study was coordinated by S.F, S.R, S.G, P.B, C.K, S.K, A.J, C.S, J.R, I.A, S.D, M.A, and V.H. All authors were involved in critical review of the manuscript. Click here for additional data file.


Parkinson's disease is a progressive neurodegenerative disease which causes health problem that affects more patients in the past few years. To be able to offer appropriate care, epidemiological analyses are crucial at the national level and its comparison with the international situation.The demographic description of reported patients with parkinsonism  according to the International Classification of Diseases (ICD-10) from the national health registries.Retrospective analysis of data available from the National Health Information System–NHIS and the National Registry of Reimbursed Health Services (NRRHS). Analyzed epidemiological data are intending to determine the regional and specific prevalence of Parkinsonism in the Czech Republic. The International Classification of Diseases diagnoses (ICD-10) of G20 (Parkinson’s disease—PD) and G23.1, G23.2, G23.3 , and G31.8  from the period of 2012 to 2018 were included into the analysis.We identified 78 453 unique patients from national registries in the period 2012 to 2018. Diagnoses of G20, G23.1, G23.2, and G31.8 were registered as the principal diagnoses in 76.6% of all individual patients.We have found a growing number of patients coded with ICD-10 of dg. G20, G23.1, G23.2, G23.3, or G31.8 . We have proven regional differences in the prevalence of Parkinson´s diagnoses. Therefore we assume most likely also differences in the care of patients with PD based on the availability of specialty care centers. Parkinson's disease (PD) is a progressive neurodegenerative disease that affects mainly the function of basal ganglia. Common PD motor signs include bradykinesia, rigidity, tremor, and postural instability. Incidence and prevalence of PD appear to increase over the recent years . PD signThe prevalence of the disease ranges from 1 to 3 per 1000 in unselected populations, and it affects 1% of the population above 60 years . AccordiÚstav zdravotnických informací a statistiky České republiky) from 2012 report 26 680 patients . The popThe demographic description of reported patients with parkinsonism  according to the International Classification of Diseases (ICD-10) from the national health registries.We performed a retrospective analysis of data available from NHIS , respectively NRRHS  with respect to the STROND checklist. In both registries, the data from health care providers about the type and amount of care are collected. We analyzed epidemiological data intending to determine the regional and specific prevalence of Parkinsonism in the Czech Republic. The International Classification of Diseases (ICD-10) diagnoses of G20 (Parkinson’s disease) and G23.1, G23.2, G23.3 , and G31.8  from the period of 2012 to 2018 were included into the analysis. Atypical parkinsonian syndromes were included due to overlap of clinical signs, at least during the initial stages of Parkinsonism. We selected the period till 2018, as the last previous data on national prevalence were published in 2018. The diagnosis G23.3 (according to ICD-10) was not found in the analyzed data, so we excluded this diagnose from our further evaluation. The standardized annual prevalence of the patients with parkinsonism  has been counted. The analyzes of age and gender distribution, age in the date of diagnosis, and regional distribution of patients within the Czech Republic districts in the analyzed sample.No patients were involved in the design of the study. For the research purposes analyses, there is not special ethical approval needed according to the Czech law if they are analyzing by the Institute of Health Information and Statistics and not by the third party.We identified 205 490 records of patients with PD from national registries in the period from 2012 to 2018  of these patients were followed under ICD-10 diagnostic codes of atypical Parkinsonism . According to available published data, the reported prevalence of atypical Parkinsonism is 0.4% (400 cases per 100 000 populations) in dementia with Lewy bodies (G31.8), multisystem atrophy with dominant parkinsonian features , and progressive supranuclear palsy  in 5 to 10 cases per 100 000 of the general population, increasing to 7 to 8 per 100 000 inhabitants in elderly . The ratAccording to data available from health registries (NHIS), we have shown that most patients diagnosed and treated with PD were in the age group of 70–84 years . Age at the time of PD diagnosis was 70–84 years, which differs from available EU data , 18. EurWe are aware of several limitations of this study. Both unintentional and intentional coder errors, such as misspecification, and up-coding, as potential sources of errors, were described . As we hThe importance of the availability of movement disorder centers is underscored by data showing that nearly half (47%) of PD diagnoses are incorrect when performed in the primary care setting. Specialists without expertise in movement disorders have an error rate of approximately 25%, while movement disorder specialists made mistakes in only 6% to 8% of cases , 27.According to available epidemiological data from the Czech health care registries, we were able to perform an analysis of population data concerning ICD-10 codes coding Parkinsonism and related disorders. We also analyzed the regional prevalence of these diagnoses in respective regions of the Czech Republic. We have found a growing number of records coded with ICD-10 of dg. G20, G23.1, G23.2, G23.3, or G31.8 . We have proven regional differences in the prevalence of diagnoses. Therefore we assume most likely also differences in the care of patients with PD based on the availability of specialty care centers. The big issue for the future in the Czech Republic is the implementation of the standardized processes of care based on nationally accepted evidence-based guidelines. 27 Nov 2020PONE-D-20-31004Representation of Parkinson's disease and atypical Parkinson's syndromes in the Czech Republic - A nationwide retrospective studyPLOS ONEDear Dr. Búřil,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Jan 11 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Weidong LeAcademic EditorPLOS ONEJournal Requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf2.Thank you for stating the following financial disclosure: .At this time, please address the following queries:Please clarify the sources of funding  for your study. List the grants or organizations that supported your study, including funding received from your institution.State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”If any authors received a salary from any of your funders, please state which authors and which funders.If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”Please include your amended statements within your cover letter; we will change the online submission form on your behalf.http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.3.We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see In your revised cover letter, please address the following prompts:a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail  and who has imposed them . Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see We will update your Data Availability statement on your behalf to reflect the information you provide.http://journals.plos.org/plosone/s/licenses-and-copyright.4.We note that [Figure(s) 3] in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software . For more information, see our copyright guidelines: We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:1.    You may seek permission from the original copyright holder of Figure(s) [3] to publish the content specifically under the CC BY 4.0 license. http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:We recommend that you contact the original copyright holder with the Content Permission Form (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0  remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.The following resources for replacing copyrighted map figures may be helpful:http://viewer.nationalmap.gov/viewer/USGS National Map Viewer (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/The Gateway to Astronaut Photography of Earth (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.htmlMaps at the CIA (public domain): http://earthobservatory.nasa.gov/NASA Earth Observatory (public domain): http://landsat.visibleearth.nasa.gov/Landsat: http://eros.usgs.gov/#USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://www.naturalearthdata.com/Natural Earth (public domain): [Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't KnowReviewer #2: I Don't Know**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In this manuscript, the authors reported a retrospective analysis of data available from the National Health Information System – NHIS and the National Registry of Reimbursed Health Services (NRRHS) with respect to the STROND checklist to determine the prevalence and other epidemiological features of Parkinsonism in the Czech Republic. Data on parkinsonian syndromes  in different Eastern European countries are scarce and this study could be valuable by depicting epidemiological features of these diseases in understudied populations. However, some changes should be reconsidered.Abstract:The aim of the study is not well stated.The results are not well defined  diagnoses of G20 (Parkinson’s disease) and G23.1, G23.2, G23.3 , and G31.8 "-How do the authors explain the fact the diagnosis G23.3 (according to ICD-10) was not found in the analyzed data? and why did they decide to excluded this diagnosis from further evaluation? This is a result they could discuss later on in the discussion part.-What do the authors mean by " regional" and "specific" prevalence of Parkinsonism. This should be better specified in the Methods part.-What do the authors mean by: " Atypical parkinsonian syndromes were 36 included due to overlap of clinical signs, at least during the initial stages of Parkinsonism."? How does this affect their methodology? this should be better explained.-The authors should precise all the data analyzed later in the results in the methodology part.Results:-What do the authors mean by "we identified 78 453 patients" and then "were registered as the principal diagnoses in 76.6 % of all individual patients"? What does the first figure stand for? all patients with "any disease"? what does the percentage stand for?-The authors stated at the end of this section: "Various genetic and social factors may influence the regional distribution of patients with Parkinsonism, as was already reported in the eastern part of our country. [13]". This should be rather put in the discussion section and better explained.-In the sentence: " The ratio of the parkinsonian subtype of PSP (PSP-P) to the cerebellar subtype is 2:1 to 4:1 in 66 most countries. ", the authors are rather talking about MSA and not PSP. This should be amended.-Other minor changes suggested:• The quality of figures and tables should beReviewer #2: In this manuscript, the authors attempted to determine epidemiological features of parkinsonian syndromes in the Czech Republic by a retrospective analysis of data available from the NHIS and the NRRHS. Further studies, such this one, on epidemiology of parkinsonism especially atypical parkinsonian syndromes are needed. However the authors should clarify some points, as follows:1-The aim/objectives of the study is not well stated in the abstract and later in the introduction.2- The authors should explain the exact meaning of the different codes used in the methodology part and precise all items/parameters that will be later detailed in the results3-The results are not well defined both in the abstract and in the manuscript4-The sentence "Various genetic and social factors may influence the regional distribution of patients with Parkinsonism, as was already reported in the eastern part of our country. [13]" should be in the discussion section.5-The quality of figures and tables could be improved.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  10 Dec 2020The aim of the study is not well stated. Reviewer #1 The aim was stated: Aim of the analyses: The main aim of our analyses was to identify the number of reported patients with Parkinson's disease (PD) – dg. G-20 (ICD-10) from the national health registries.The results are not well defined  diagnoses of G20 (Parkinson’s disease) and G23.1, G23.2, G23.3 , and G31.8 " Reviewer #1 Accepted – the data were analysed based on the record and codes reported by clinicians to the National health registries.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0198736&type=printable). How do the authors explain the fact the diagnosis G23.3 (according to ICD-10) was not found in the analyzed data? and why did they decide to excluded this diagnosis from further evaluation? This is a result they could discuss later on in the discussion part. Reviewer #1 Accepted – the diagnosis G23.3 (according to ICD-10) was not found in the analyzed data – there were no record of the dg. G23.3 in the whole analysed dataset. The possible reason is, that there were no incidence of patients with dg. G23.3 which could relate to the problem that the clinicians are unable to diagnose it appropriately. Also published literature is confirming that the dg. G23.3 is not reported, the commonly used codes are 23.1, 23.2 and 23.9 , which means 78 453 unique patients in total . We have found a growing number of records coded with ICD-10 of dg. G20, G23.1, G23.2, or G31.8 .The authors stated at the end of this section: "Various genetic and social factors may influence the regional distribution of patients with Parkinsonism, as was already reported in the eastern part of our country. [13]". This should be rather put in the discussion section and better explained. Reviewer #1 Accepted - we add the information in the discussion section.In the sentence: "The ratio of the parkinsonian subtype of PSP (PSP-P) to the cerebellar subtype is 2:1 to 4:1 in 66 most countries. ", the authors are rather talking about MSA and not PSP. This should be amended. Reviewer #1 Accepted - sorry for the misinterpretation – the text was changed. Other minor changes suggested:• The quality of figures and tables should be Reviewer #1 We could not address this comment – maybe the comment was not finished?The aim/objectives of the study is not well stated in the abstract and later in the introduction. Reviewer #2 The aim was stated: Aim of the analyses: The demographic description of reported patients with Parkinson's disease according to the International Classification of Diseases (ICD-10) from the national health registries. .The authors should explain the exact meaning of the different codes used in the methodology part and precise all items/parameters that will be later detailed in the results Reviewer #2 Accepted – double checked and added explanations.The results are not well defined both in the abstract and in the manuscript Reviewer #2 Accepted – double checked and added explanations.The sentence "Various genetic and social factors may influence the regional distribution of patients with Parkinsonism, as was already reported in the eastern part of our country. [13]" should be in the discussion section. Reviewer #2 Accepted - we add the information in the discussion section.The quality of figures and tables could be improved. Reviewer #2 The figures are used from the data analyses system – we have no possibility to improve the quality.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Editor Accepted – Thank you so much.At this time, please address the following queries:a) Please clarify the sources of funding  for your study. List the grants or organizations that supported your study, including funding received from your institution.b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”c) If any authors received a salary from any of your funders, please state which authors and which funders.d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Editor The authors received no specific funding for this work.http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see In your revised cover letter, please address the following prompts:a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail  and who has imposed them . Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see Jiri.Jarkovsky@uzis.cz)We will update your Data Availability statement on your behalf to reflect the information you provide. Editor There is legal restrictions on sharing data publicly – all the data form the National registries must be kept safely and authors are working with the anonymised data. For the data you can ask the head of the department of data Analyses team from the Institute of Health Information and Statistics of the Czech Republic - Dr. Jiří Jarkovský  3] in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software . For more information, see our copyright guidelines: We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:1. You may seek permission from the original copyright holder of Figure(s) [3] to publish the content specifically under the CC BY 4.0 license. http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:We recommend that you contact the original copyright holder with the Content Permission Form (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0  remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.The following resources for replacing copyrighted map figures may be helpful... Editor The figure no. 3 was produced by our author’s team. We have published this kind of figure with different type of health data has been already published without any need for copyrighted.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 30 Dec 2020PONE-D-20-31004R1Representation of Parkinson's disease and atypical Parkinson's syndromes in the Czech Republic - A nationwide retrospective studyPLOS ONEDear Dr. Búřil,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Feb 13 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Weidong LeAcademic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: NoReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Most of the required modifications were amended. However, minor revisions are still needed:1-In the abstract and in the manuscript: in the aim of the study, " The demographic description of reported patients with Parkinson's disease"==> The demographic description of reported patients with parkinsonism : the aim should be in accordance with the article title.2-The authors should precise all the data analyzed later in the results in the methodology part. This was not amended.3-The quality of figures and tables should be improved.Reviewer #2: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  6 Jan 2021In the abstract and in the manuscript: in the aim of the study, " The demographic description of reported patients with Parkinson's disease"==> The demographic description of reported patients with parkinsonism : the aim should be in accordance with the article title. Accepted – the aim has been reworded according the reviewer´s comment.The authors should precise all the data analyzed later in the results in the methodology part. This was not amended. Accepted – the information in the methodology part has been added.The quality of figures and tables should be improved. Accepted – the figures were graphically edited and uploaded.AttachmentResponse to Reviewers_6_1_2021.docxSubmitted filename: Click here for additional data file. 18 Jan 2021Representation of Parkinson's disease and atypical Parkinson's syndromes in the Czech Republic - A nationwide retrospective studyPONE-D-20-31004R2Dear Dr. Búřil,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Weidong LeAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: No 21 Jan 2021PONE-D-20-31004R2 Representation of Parkinson's disease and atypical Parkinson's syndromesin the Czech Republic - A nationwide retrospective study Dear Dr. Búřil:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Weidong Le Academic EditorPLOS ONE